To study the extraordinary complexity of biological systems in a relevant way requires both multidimensional and highly specific analyses. Ultimately, I want to build a chemical biology research program using synthetic protein chemistry and a systems biology approach to address the challenges of studying complexity in a simple way. I plan to use chemical protein synthesis as a combinatorial strategy to generate molecular biosensors with enough features to define differences and signatures in kinase signaling pathways related to breast cancer, and that also allow for the incorporation of chemical tricks to improve signal to noise and ease analysis. The biosensors will have clinical applications in detecting, monitoring and designing treatment for cancer-related pathways, and will provide unique information about the relevance of interrelated kinase activities to cancer disease states with high signal to noise, resolution and specificity. The specific aims are to: build and characterize an initial library of artificial, targeted kinase substrate proteins; use these artificial substrates for in vivo detection of cancer-related kinase activities; and use the in vivo phosphorylation information in a systems-level approach to define cancer-related multi-substrate 'signatures' to serve as biosensors for disease states. To generate these biosensors, small peptide units with different specific functions will be synthesized using solid-phase peptide synthesis and linked to each other using native chemical ligation to make small (100-150 aa) artificial proteins. These proteins will consist of modules for directing their cellular internalization and localization, enhancing specific protein-protein interactions, reporting their location via fluorescence and acting as kinase substrates for particular signaling pathways. The kinase substrate portion will be selectively releasable, and contain an affinity tag for rapid purification of this segment and analysis using MALDI-TOF mass spectrometry as a reporter for kinase activity. In this project, artificial protein biosensors will be designed to help us visualize the complicated patterns of abnormal tyrosine kinase activity in diseases like breast cancer. In many breast cancers, these tyrosine kinases are highly abundant and overactive, disrupting the normal cellular processes that keep growth from getting out of control. Understanding how their activities interrelate and the timing of changes in their patterns will help us better diagnose and treat diseases that show these characteristic complexities.